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Results: 5

1.
Fig. 3

Fig. 3. From: Context-dependent enhancement of induced pluripotent stem cell reprogramming by silencing Puma.

Puma and p21 activities cooperatively suppress induced pluripotency. Wild-type, Puma−/−, p21−/−, Puma−/−/p21−/− and p53−/− MEFs (p6) were transduced with Oct3/4-, Sox2-, Klf4-expressing retrovirus, and analyzed for Alkaline Phosphatase (AP) staining after two weeks. Reprogramming efficiency was determined relative to the wild-type condition and shown as mean ± s.d. (N=3).

Blue B. Lake, et al. Stem Cells. ;30(5):888-897.
2.
Fig. 2

Fig. 2. From: Context-dependent enhancement of induced pluripotent stem cell reprogramming by silencing Puma.

Reprogramming Efficiency is limited by both p53-dependent cell cycle arrest and apoptosis. Wild-type, p53S18A, p53S18/23A, Puma−/−, p21−/− and p53−/− MEFs were transduced with Oct3/4, Sox2, Klf4 retroviruses with (A) or without (B) c-Myc and analyzed for Alkaline Phosphatase (AP) staining (C) after two weeks. MEFs carrying humanized knock-in p53 allelle (HKI) with or without point mutation at Ser46 (HKIp53S46A, D) were analyzed for AP staining following 3 factor transduction. All analyses are relative to the wild-type condition (N=3) and are mean ± s.d. Total number of SSEA-1 positive cells was determined through FACs analysis of day 10 and day 14 three factor reprogramming cultures (E).

Blue B. Lake, et al. Stem Cells. ;30(5):888-897.
3.
Fig. 4

Fig. 4. From: Context-dependent enhancement of induced pluripotent stem cell reprogramming by silencing Puma.

Puma is not required to suppress DNA damage during reprogramming. A. Confocal analysis of day 14 SSEA-1 positive colonies for γH2AX and DAPI. B. FACS analyses of day 14 to 18 OSK(M) cultures co-stained with γH2AX and SSEA-1. (Wild-type, Wild-type + c-Myc, p53S18A, p53S18/23A, Puma−/−/p21−/−: N=3; Puma−/−, p21−/−, p53−/−: N=6). Values are mean ± s.d. Significant differences (*p<0.05; **p<0.01) are with respect to p53−/− unless otherwise indicated. C. Cell cycle analysis of Puma−/− and p53−/− MEFs compared to wild-type MEFs after 7 days of OSK reprogramming through propidium iodide (PI) staining and flow cytometry (N=6). Percentage of G2/M fraction showing phospho-histone H3 (Ser10) positive staining relative to wild-type as determined by flow cytometry with values shown as mean ± s.d. (N=3). D. β-galactosidase staining at day 11 following OSK infection and corresponding relative quantification of positive cells.

Blue B. Lake, et al. Stem Cells. ;30(5):888-897.
4.
Fig. 5

Fig. 5. From: Context-dependent enhancement of induced pluripotent stem cell reprogramming by silencing Puma.

Loss of Puma and p21 permits generation of stable mouse iPSCs. A. Representative iPSCs (passage 5-6) from each genotype stained for alkaline phosphatase (AP) or SSEA-1 and Oct3/4. B. Real time PCR analysis of pluripotency marker gene expression relative to wildtype MEFs. Mouse iPSCs (passage 5-6) were averaged within genotype (Wild-type, N=2; Puma/p21 DKO, N=3; p53−/−, N=2) and compared with J1 ES cells. C. Hematoxylin-Eosin staining of Puma/p21 DKO teratoma sections showing representative mesodermal (muscle, cartilage), endodermal (glands, gut-like epithelium) and ectodermal (epidermis, neurepithelium) tissues. D. Adult chimeric mouse generated from injection of Puma/p21 DKO iPSCs into an albino blastocyst. E. Flow cytometric analysis of SSEA-1 in representative early passage (p3-4) and late passage (p23-24) murine iPS cell lines compared to J1 ESCs. F. Theoretical scheme for p53 pathway response to DNA damage during OSK reprogramming of wildtype or Puma−/− (G) MEFs. OIS, oncogene-induced stress; RS, replicative stress; DD, DNA damage.

Blue B. Lake, et al. Stem Cells. ;30(5):888-897.
5.
Fig. 1

Fig. 1. From: Context-dependent enhancement of induced pluripotent stem cell reprogramming by silencing Puma.

p53-dependent DNA damage response is activated during reprogramming. A. Real-time PCR analysis of p53 target gene expression at progressive time-points of three factor reprogramming of Wild-type MEFs. Results are shown relative to Wild-type uninfected MEFs collected at the start of each experiment (N=3). B. Respective level of reprogramming at the associated time-points was indicated by nanog gene expression relative to untransduced wild-type MEFs (N=3). C. Real-time PCR analysis of p53 target gene expression in p53S18A, p53S18/23A, Puma−/−, p21−/− and p53−/− MEFs on days 3 (N=2) and 7 (N=3) of three factor reprogramming. Results are shown relative to wild-type (OSK) for each respective time-point and are mean ± s.d. Confocal analysis of γH2AX and p-ATM (D, arrows) or p-p53(Ser18) and p-ATM (E, arrows) immunoflourescent staining of wild-type MEFs on day 7 of reprogramming (OSK). F. Quantification of γH2AX/p-ATM positive foci in Wild-type, Puma−/− and p53−/− day 7 reprogramming (OSK) cultures relative to uninfected wild-type MEFs at the same time-point. Results are over 10 fields (40x objective) per experiment and shown as mean ± s.d. between experiments (N=2). G. Western blot analyses of Day 11 reprogramming (OSK) cultures (treated on Day 10 with 20mJ UV) for Cleaved Caspase 3 and α-Tubulin (N=2). H. Apoptosis on Day 11 of reprogramming (OSK) was determined in UV treated cultures (20mJ applied on Day 10) through co-staining for active caspases (FAM-VAD-FMK) and Thy1. Percentage of cells showing general caspase activity within the Thy1+ or Thy1 fractions are shown as percentages relative to Wild-type (OSK) cultures untreated for UV and are shown as mean ± s.d. (N=3). **p<0.01

Blue B. Lake, et al. Stem Cells. ;30(5):888-897.

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